JP2003107090A - Labelling complex composition for immune chromatograph - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a labelling complex composition for immune chromatograph with reliability in detecting a substance to be detected within a wide range in the amount of the substance to be detected and immune chromatography using the same. SOLUTION: The labelling complex composition includes two types or more of labelling complex each with a labeling substance and an antibody to the substance to be detected, and the detection sensitivity of the antibody in each labeling complex to the substance to be detected is different from each other in the labelling complex composition for immune chromatograph. Through the use of a test piece with a solid phase in which the antibody to the substance to be detected is immobilized on an water absorptive substance, the presence or absence of the substance to be detected in a sample to be tested is detected by the presence or absence of a complex between (a) and (b) (wherein, (a) is the substance to be detected, and (b) is the labelling complex in the labelling complex composition mentioned in any of Claims 1-4) in the immune chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a labeled complex composition for immunochromatography having high certainty in detecting a wide range of amounts of a test substance, and an immunochromatographic method using the same.

[0002]

2. Description of the Related Art In recent years, an immunochromatographic method has been attracting attention as a method capable of conducting an immunochemical test quickly and easily. The method includes, for example, the following steps. When a test substance is present in a test sample, a specific binding substance capable of specifically binding to the test substance is applied to a stationary phase on which a specific binding substance capable of binding to the test substance is immobilized on a test piece. A complex of a labeled complex containing the and the labeled substance and the test substance is formed. Then, the presence of the test substance in the test sample can be confirmed by detecting the labeled substance bound in the stationary phase.

However, when the test substance is present in excess, after the complex of the labeled complex and the test substance is formed, the obtained complexes become aggregates, and the chromatographic expansion proceeds normally. There are times when you can't forget. Usually, the immunochromatography method is provided with a reference line, and it is confirmed that the test is normally performed by capturing the unreacted labeled complex. However, many chromatographic kits use an immunoglobulin-labeled substance of an unsensitized animal to determine the success or failure of the test. This involves the problem that it cannot be the direct evidence that the labeled complex to the actual test substance is normally developed.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a labeled complex composition for immunochromatography having certainty in detecting a test substance in a wide range of the amount of the test substance, and an immunochromatographic method using the same. To provide.

[0005]

That is, the gist of the present invention is [1] a labeled complex composition containing two or more types of labeled complexes having a labeled substance and an antibody against a test substance,
The labeled complex composition for immunochromatography, wherein the detection sensitivities of the antibodies in each labeled complex to the test substance are different from each other, and [2] on a water-absorbent substrate,
The presence or absence of the test substance in the test sample is determined by using a test piece having a stationary phase in which an antibody against the test substance is immobilized.
The following (a) and (b): (a) a test substance, (b) the labeled complex in the labeled complex composition described in [1] above, and the detection is performed depending on the presence or absence of a complex. , Immunochromatography.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The labeled complex composition for immunochromatography of the present invention is a labeled complex composition containing two or more types of labeled complexes having a labeling substance and an antibody against a test substance. One feature is that the detection sensitivities of the antibodies in the labeled complex to the test substance are different from each other.
By carrying out an immunochromatographic method using the labeled complex composition of the present invention, it is possible to detect the test substance even if the amount of the test substance is small, and conversely, even if the amount of the test substance is large, the labeled complex is detected. Since the body develops well, the test substance can be detected with high reliability in a wide range of the test substance amount.

The difference between the maximum detection sensitivity and the minimum detection sensitivity of the antibody against the test substance is measured by immunochromatography,
The difference is preferably 5 to 5 × 10 ⁴ times, more preferably 50 to 1 × 10 ³ times. Antibodies having different detection sensitivities to the test substance are selected, for example, by the following method. As the antibody to the test substance, several types can be obtained by a commonly used method, such as a polyclonal antibody or a monoclonal antibody. Each antibody is bound to a labeling substance to prepare a labeling complex of the antibody whose sensitivity is to be measured. Chromatographic test paper is produced by a conventional method using a nitrocellulose membrane. For the measurement, a sandwich method is used, and a fixed amount of an antibody against the test substance (reference antibody) is applied to the test paper side to form a detection line. Using this chromatographic test paper, an immunochromatography kit in which a sample dropping part, a water absorbing material and the like are arranged is produced. Using the immunochromatography kit thus prepared and the labeled complex, a serially diluted solution of the test substance (for example, a 10-fold dilution series), that is, a standard solution is measured, and as a result, the test substance for each antibody is tested. The minimum detectable concentration is determined as the detection sensitivity. In this way, antibodies with different detection sensitivities can be selected. For example, when the test substance is Escherichia coli O157, E. coli O1
As a sample with the lowest concentration capable of detecting 57, for example, 10 ⁴
The sensitivity of the antibody that can be detected from the sample containing 10 cells / mL is 10 ⁴ cells / mL, and the sensitivity of the antibody that can be detected from the sample containing the minimum concentration of 10 ⁶ cells / mL is 10 ⁶ cells / mL. Therefore, in this case, there is a 100-fold difference in detection sensitivity between the two antibodies.

[0008] The two or more kinds of antibodies having different detection sensitivities to the test substance may be derived from different animals or the same species. Further, it may be either a monoclonal antibody or a polyclonal antibody.

The content of the labeled complex containing the antibody having the maximum detection sensitivity for the test substance is such that the low concentration of the test substance is detected with high sensitivity and the unbound labeled complex reaches the reference phase. From the viewpoint that it is possible, 30 to 95% by weight of the labeled complex composition is preferable, and 50 to 8%.
0% by weight is more preferred.

The content rate of the labeled complex containing the antibody having the minimum detection sensitivity to the test substance is not taken into the aggregate when the test substance is present in a large excess, and the labeled complex can be developed by chromatography. 5 to 70% by weight of the composition is preferable, and 20 to 50% by weight is more preferable.

By using a labeled complex containing an antibody having a high detection sensitivity for a test substance, a low-concentration test substance can be detected with higher sensitivity.

Further, by using a labeled complex containing an antibody having a low detection sensitivity for the test substance, the labeled complex containing an antibody having a low detection sensitivity can be aggregated even when the test substance is present in a large excess. Chromatographic development is possible without forming

The test substance that can be detected by the immunochromatographic method of the present invention is not particularly limited as long as it can form a sandwich immune complex by an immunochemical reaction (that is, an antigen-antibody reaction). Examples thereof include bacteria such as Escherichia coli and its constituents, toxins produced by bacteria, proteins, viral antigens and antibodies, biologically-derived components such as Mycoplasma, and endocrine disruptors, pesticides, chemicals such as pharmaceuticals.

In the labeling complex having a labeling substance and an antibody, examples of the labeling substance include known substances used in the immunoassay field, such as colored particles, enzymes and fluorescent substances. Among them, colored particles and enzymes are more preferable because they can be visually determined and a measuring device is not required.

The colored particles may be those whose color can be detected with the naked eye, for example, colored with dyes or pigments represented by Sudan Blue, Sudan Red IV, Sudan III, Oil Orange, Quinizarine Green and the like. Examples thereof include water-dispersed polymer particles. From the viewpoint of visual confirmation, colored water-dispersed polymer particles colored in blue, red, green or orange are preferable. Colored latex particles colored in blue or red are desirable from the viewpoint of easy adjustment of dispersion stability and detection sensitivity of a test substance. The average particle diameter of the colored particles is about 0.01 μm or more, from the viewpoint of good color development.
It is preferably 0.05 μm or more, and from the viewpoint of preventing clogging of the water-absorbent substrate due to slight aggregation of the colored particles,
It is desirable that the thickness is about 3 μm or less, preferably about 0.5 μm or less. Specifically, it is desired to be in the range of about 0.01 to 3 μm, preferably about 0.05 to 0.5 μm.

Examples of the enzyme include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxidase, luciferase, esterase, β-D-glucuronidase and the like. Peroxidase or alkaline phosphatase, which are capable of achieving more sensitive and stable detection, are preferred.

Examples of the fluorescent substance include fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.

Further, in the present invention, the labeling complex uses metal colloid particles or water-dispersed polymer particles as a carrier, and the labeling substance (the enzyme or fluorescent substance) and the antibody are immobilized on the carrier. It may be a complex.

The carrier used in the labeling complex may be any one that can immobilize the antibody and the labeling substance on the surface thereof, such as metal colloid particles, water-dispersible polymer particles, silicone, silica and glass. Examples include diatomaceous earth particles.

Examples of the metal colloid particles include gold colloid particles and selenium colloid particles.

As the water-dispersible polymer particles, latex particles are preferable from the viewpoints of particle size control, dispersion stability and easiness of binding. The water-dispersible polymer particles are prepared, for example, by emulsion polymerization of at least one monomer having an unsaturated double bond. Examples of such monomers include ethylene, olefinic monomers such as propylene, vinyl acetate, vinylic monomers such as vinyl chloride, styrene,
Examples thereof include styrene-based monomers such as methylstyrene and chlorostyrene, methacrylic acid ester-based monomers such as methylmethacrylate, and diene-based monomers such as butadiene.

The water-dispersible polymer particles may be a polymer obtained by copolymerizing a monomer homopolymer or copolymer with a modifying monomer. Examples of the modifying monomer include acrylic acid, methacrylic acid, fluorinated methacrylic acid esters such as 2,2,2-trifluoroethylmethyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide, and styrene sulfonic acid. sodium,
Sulfopropyl (meth) acrylate sodium salt, N
-Vinyl-2-pyrrolidone, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate and the like can be mentioned.

In the present invention, the carrier may be various commercially available water-dispersible polymer particles. Examples of commercially available water-dispersible polymer particles include styrene-
Emulsions of homopolymers or copolymers containing a styrene-containing material or its derivative as a monomer component, such as emulsions of various styrene copolymers such as butadiene copolymers and styrene-acrylonitrile-butadiene copolymers;
A homopolymer containing a long-chain alkyl ester of (meth) acrylic acid or a derivative thereof as a monomer component, the monomer component and methyl (meth) acrylate, ethyl (meth) acrylate, glycidyl (meth) acrylate, etc. A copolymer with
Examples thereof include copolymers of the above-mentioned styrene or a derivative thereof and a (meth) acrylate ester or a derivative thereof; rubber, nylon, polyurethane, microcrystalline cellulose and the like.

The specific type of each monomer is the above-mentioned aqueous dispersion so that the antibody using the resulting water-dispersible polymer particles as a carrier does not cause fusion or aggregation during use or storage. The polymeric particles of type are selected to have the required glass transition point. The glass transition point of the water-dispersed polymer particles is preferably 10 ° C. or higher, and particularly preferably room temperature or higher.

Among the water-dispersible polymer particles, a polymer containing a styrene-based monomer as a main component is preferable from the viewpoint of particle stability, and acrylic acid is used for immobilizing the antibody and the labeling substance. Polymer particles obtained by copolymerizing a monomer having a carboxyl group such as or methacrylic acid are desirable.

The carrier may have a functional group from the viewpoint of immobilizing the antibody and the labeling substance on the surface of the carrier, introducing a spacer, or improving stability in a water dispersed state.
Examples of such a functional group include a carboxyl group, a hydroxyl group, a glycidyl group, an amino group, a formyl group, a carbamoyl group, an isothiocyanate group, an azidocarbonyl group,
Examples thereof include a hydrazide group and an acid anhydride group, and preferably a carboxyl group is introduced. To prepare a carrier having these functional groups, as a monomer component, for example, a monomer having a carboxyl group such as acrylic acid or methacrylic acid, such as hydroxyethyl acrylate or 2-hydroxyethyl methacrylate. Monomers having a hydroxyl group, for example, by copolymerizing a monomer having a glycidyl group such as glycidyl methacrylate, or by emulsion copolymerization with other copolymerizable monomers as necessary, respectively carboxyl A carrier having a group, a hydroxyl group and a glycidyl group can be obtained. Further, after polymerizing a required monomer component, a functional group can be introduced into the obtained carrier.

The particle size of the carrier depends on the dispersibility, the enzyme,
From the viewpoint of improving the amount of immobilized antibody and the like, it is preferably 3 μm or less, more preferably 2 μm or less,
From the viewpoint of easiness of purification of the obtained labeled complex, it is desirable that it is preferably 0.01 μm or more, more preferably 0.1 μm or more.

As a method for immobilizing the antibody and the labeling substance on the carrier, hydrophobic bond (physical adsorption), ionic bond,
A covalent bond or the like can be used. From the viewpoint of stability, when binding via a covalent bond, a spacer group may be interposed, if necessary, in order to increase the degree of freedom on the water-dispersed polymer particles of the antibody or the like.

The spacer group may be previously bound to the water-dispersible polymer particles and then bound to the antibody and the labeling substance. Alternatively, the spacer group may be bound to the antibody and the labeling substance in advance and then the water-dispersible polymer is used. It may be bound to the particles. Furthermore, if necessary, a spacer group may be previously bound to all of the water-dispersed polymer particles, the antibody and the labeling substance, and these may be bound to each other.

The compound that can be used as the spacer group is at least a bifunctional organic compound, and particularly has a carbon number of 1 to 1.
Bifunctional organic compounds having 12 carbon chain groups are preferred. The compound functioning as such a spacer group is not particularly limited, but for example, hexamethylenediamine, dodecamethylenediamine, diamines such as xylylenediamine, glycine, β-aminopropionic acid,
Aminoalkylcarboxylic acids such as γ-aminobutyric acid, ε-aminocaproic acid and ε-aminocaprylic acid, amino acids such as lysine, glutamic acid, β-alanine, arginine and glycylglycine are preferable.

The method for covalently bonding the antibody and the labeling substance to the water-dispersed polymer particles directly or via a spacer group is not particularly limited, and a conventional method can be mentioned. Examples of such a method include a method using a water-soluble carbodiimide as a binding reagent. Specifically, for example, when aminoalkylcarboxylic acid is used as the spacer group, the water-soluble carbodiimide can be used to bind the antibody and the labeling substance to the water-dispersed polymer particles by a covalent bond.

Examples of the water-soluble carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl)
Carbodiimide hydrochloride, 1-cyclohexyl-3- (2
-Morpholinoethyl) carbodiimide-meth-p-toluenesulfonate and the like. Coupling of the antibody and the labeling substance to the water-dispersible polymer particles through a spacer group or by a direct covalent bond using such a water-soluble carbodiimide can be performed by a conventional method and conditions. In the present invention, the antibody and the labeling substance may be bound to the water-dispersible polymer particles either at the same time or separately.

The total amount of the antibody and the labeling substance contained in the labeling complex thus obtained is preferably 5 to 200 mg per 1 g of the dry weight of the carrier, and the amount is within the above range. It may be appropriately changed depending on the type of antibody, labeling substance, etc. to be used. For example, when the carrier is water-dispersed polymer particles, in view of the surface area of the particles, the total fixed amount is preferably 200 mg or less, more preferably 150 m, per 1 g of the dry weight of the water-dispersed polymer particles.
It is preferably g or less, more preferably 5 mg or more, and further preferably 10 mg or more, from the viewpoint of the rapidity of detection of the test substance, sensitivity, and reproducibility.

Here, the "dry weight" of the carrier means the weight after drying a fixed amount of the carrier at 120 ° C for 2 hours.

The immunochromatographic method includes, for example, a method including the following steps. That is, from one end of a test piece having a stationary phase in which an antibody against a test substance is immobilized on a water-absorbent substrate, a solution containing a labeled complex composition and a test sample are separately or Are mixed in advance and dropped on a test piece to be developed. When the test substance is present in the test sample, the complex composed of [test substance-labeled complex] is bound to the antibody immobilized on the stationary phase and captured on the stationary phase. . Therefore, the test substance in the test sample can be measured by measuring the labeled substance in the complex composed of [test substance-labeled complex] captured on the stationary phase.

Examples of the test sample include a sample suspected of containing a test substance. Specifically, blood components,
Examples thereof include biologically derived components such as urine, saliva, sweat and stool, foods and culture solutions. The test sample may be a liquid sample or a solid sample. For solid samples,
The solid sample may be used as a solution obtained by dissolving or diluting it in a solvent such as a buffer solution or physiological saline.

The water-absorbent substrate used for the test piece may be any substrate capable of absorbing the test sample or a substrate absorbing a diluting solution obtained by diluting these with a buffer solution. In the present invention, a water-absorbent substrate is used that can secure a sufficient time for sufficient reaction between the test substance in the test sample and the antibody in the labeled complex or the antibody in the stationary phase. As a preferred specific example, from the viewpoint of having an appropriate water absorption rate, for example,
Nonwoven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose, porous material and the like can be mentioned.

The degree of water absorption of the water-absorbent substrate is determined by immersing water in one end of the water-absorbent substrate cut into strips each having a width of 5 mm.
The water absorption distance after 0.5 minutes is preferably 0.5 to 5 cm. A hydrophilic polymer can also be used to adjust the water absorption of the water-absorbent substrate.

In the present invention, the shape of the water-absorbent substrate is not particularly limited as long as the test sample can be developed, and for example, a rectangular sheet shape (piece shape) or a rod shape is preferable.

As the antibody to the test substance used in the stationary phase of the test piece, the same antibody as the antibody used in the labeled complex or an antibody recognizing another epitope of the same antigen can be used.

The stationary phase can be prepared by a known physical adsorption method, covalent bonding method or the like. Further, after applying a solution containing an antibody used in the stationary phase and a hydrophilic polymer to the water-absorbent substrate,
The stationary phase can also be prepared by immersing the hydrophilic polymer in a coagulating solvent that coagulates the hydrophilic polymer. Examples of the hydrophilic polymer include hydroxypropylmethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose and the like. Examples of the coagulating solvent include acetone, ethanol, methanol, ether and the like.

The stationary phase captures the complex consisting of [test substance-labeled complex] that has developed and moved by the absorption of the test sample, and 0.005 to 5% of the antibody on the water-absorbent substrate.
It is desirable to apply 5 mg / cm ² .

The water-absorbent substrate after fixation has a blocking agent and a surface active agent from the viewpoints of preventing non-specific adsorption of non-test protein to the substrate, easiness of development, and storage stability of the immobilized antibody. Treatment with a solution containing an agent and sugar (referred to as a treatment liquid) is preferable. Here, examples of the blocking agent used include bovine serum albumin, casein, gelatin, skim milk and the like. As the surfactant, polyoxyethylene (10) octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether, etc. Is mentioned. The protein content in the treatment liquid is preferably 0.1 to 10% by weight. The content of the surfactant in the treatment liquid is preferably 0.01 to
It is 1% by weight. The content of sugar in the treatment liquid is 0.1 to
It is 10% by weight.

In the test piece, a reference phase on which a substance capable of specifically binding to the antibody contained in the labeled complex is immobilized may be provided on the water-absorbent substrate downstream of the stationary phase.
In the reference phase, the developed labeled complex itself can be captured. By providing the reference phase, it is possible to directly determine the success or failure of the test by the immunochromatography method.

In this specification, "downstream" means
It means the stationary phase side as seen from the test sample receiving part described later.

The substance capable of specifically binding to the antibody contained in the labeled complex used in the reference phase is, for example, an antibody against the antibody contained in the labeled complex (anti-IgG).
Antibody, anti-Fc antibody, etc.), protein A with antibody binding property
And Protein G and the like. Here, the labeled complex to be captured is, for example, an IgG derived from the same animal as the antibody species used for the labeled complex having high detection sensitivity and the labeled complex having low detection sensitivity.
, An anti-IgG antibody against the IgG of the animal can be used alone. In the case of different animals, anti-IgG antibody against the antibody derived from each animal can be mixed and used. In this way, any labeled complex can be captured.

The reference phase can be prepared by the same method as the stationary phase. In addition, the reference phase is a substance capable of specifically binding to the antibody contained in the labeled complex on the water-absorbent substrate in order to capture the labeled complex that has developed and moved by the absorption of the test sample or the like. 0.05 to
It is desirable to apply 10 μg / cm ² .

The water-absorbent substrate used in the present invention is supplied with a test sample receiving part (a part for supplying the test sample) and a labeled complex receiving part (a solution containing the labeled complex composition). Section) may be provided. When an enzyme is used as the labeling substance, a chromogenic substrate solution receiving part (a part for supplying the chromogenic substrate solution) may be further provided. The solution containing the labeled complex composition can be obtained by, for example, dissolving the labeled complex composition in a buffer solution or the like. Further, the labeled complex may be detachably retained on the test piece to provide the labeled complex phase so that the labeled complex can be developed by contact with the liquid component contained in the test sample or the like.

Further, in the test piece used in the present invention,
The development moving distance is 0.5 cm or more, preferably 1 cm or more from the viewpoint of color development uniformity and color development sensitivity in the stationary phase, and the reachability of the test sample to the stationary phase, the color development sensitivity and the measurement time. To 8 cm or less, preferably 6 cm
It is desirable that the settings are as follows.

In the immunochromatographic method of the present invention, for example, a test piece in which a labeled complex receiving part, a test sample receiving part, a stationary phase and a reference phase are arranged so as to obtain the expansion migration distance is used. it can.

As a developing method, for example, a method of adding a solution containing the labeled complex composition and a solution such as a test sample from one end side of the water-absorbent substrate and allowing the solution to spontaneously develop by a capillary phenomenon is mentioned. . Further, the water-absorbent substrate may be provided with a water-absorbing pad at the downstream end of the stationary phase,
As a result, the liquid component that develops the test piece is absorbed, so that the development easily proceeds.

A method for detecting a test substance according to the present invention will be described with reference to FIG. 1 as a typical example. Test sample receiving part 2
Then, the test sample is dropped using a pipette or the like, and the solution containing the labeled complex composition is dropped into the labeled complex receiving section 1.
The solution containing the test sample and the labeled complex composition spreads on the test piece in the direction of the water absorption pad 5, and the stationary phase 3
To move to the reference phase 4. When the test substance is present in the test sample, it is formed [test substance-
Labeled complex] is bound to the antibody against the test substance immobilized on the stationary phase 3 and captured on the stationary phase 3. That is, when the labeling substance is detected in the stationary phase 3, it serves as an indicator that the test substance is present in the test sample. Further, when the development of the labeled complex is normal, the remaining labeled complex not captured in the stationary phase 3 can specifically bind to the antibody contained in the labeled complex immobilized in the reference phase 4. It is bound to the substance and captured in the reference phase 4. That is, when the labeling substance is detected in the reference phase 4, it is an index that normal chromatographic development was performed in the test by the immunochromatography method.

[0053]

Examples Preparation Example 1: Preparation of labeled complex composition 1) Preparation of colored latex particle suspension 50 g of styrene monomer, 0.5 g of acrylic acid, 0.2 g of triethylene glycol methacrylate, and 440 g of distilled water The mixed liquid consisting of 7
An aqueous solution prepared by dissolving 0.25 g of potassium persulfate as a polymerization initiator in 10 g of distilled water was added while maintaining the temperature at 5 ° C, and polymerization was carried out for 10 hours to obtain polystyrene latex particles (average particle diameter: 0.2 μm). Got The obtained latex particles were mixed with a buffer solution (0.01 M-borate buffer solution, pH
It was dispersed in 8.2) so that the solid content concentration was 10% by weight to obtain a latex particle dispersion liquid. Sudan Blue 0.2
g was dissolved in 20 ml of toluene, 0.2 g of sodium dodecyl sulfate and 100 ml of distilled water were added, and this mixed solution was emulsified with an ultrasonic disperser. 30 ml of the latex particle dispersion liquid was added to this emulsion, and the mixture was stirred at room temperature for 24 hours. After removing toluene from the obtained solution with an evaporator, a buffer solution (0.01 M-borate buffer solution, pH
Centrifugal washing in 8.2) was carried out and dispersed so that the solid content concentration became 5% by weight to obtain a colored latex particle suspension.

2) Immobilization of antibody In this example, goat anti-E. Coli O157: H7 was used as the antibody.
The polyclonal antibody was immobilized on the colored latex particles obtained in the above 1) as follows. Two kinds of antibody-bonded colored latex particles were prepared by using two kinds of antibodies having different detection sensitivities to E. coli O157 as a test substance.

High-sensitivity antibody-bonded colored latex particles 3 ml of the colored latex particle suspension obtained in 1) above
0.5 ml of a water-soluble carbodiimide [manufactured by Dojindo Laboratories, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01 M borate buffer (pH 8.2)] And goat anti-E.coli O15
7: H7 polyclonal antibody [immunochromatographic sensitivity to Escherichia coli O157: 10 ⁴ cells / ml, Kirkegaard & Perry
Made by Laboratories Inc. (KPL), 1 mg / ml, 0.01
2 ml of M-borate buffer (pH 8.2)],
The reaction was carried out at 10 ° C for 3 hours. Then, with respect to the obtained reaction product, 0.01 M-borate buffer solution (pH
Centrifuge and wash using 8.2) to obtain 0.01M
-Prepared with borate buffer to a solid content concentration of 5% by weight to obtain a highly sensitive antibody-bound colored latex particle suspension.

Low-sensitivity antibody-bound colored latex particles: 3 ml of the colored latex particle suspension obtained in 1) above.
0.5 ml of a water-soluble carbodiimide [manufactured by Dojindo Laboratories, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / m1, 0.01 M borate buffer (pH 8.2)] And goat anti-E.coli O15
7: H7 polyclonal antibody [immunochromatographic sensitivity to Escherichia coli O157: 10 ⁶ cells / ml, manufactured by Immunoprobe,
1 mg / m1, 0.01 M borate buffer (pH 8.
2)] 2 ml was added and the mixture was reacted at 10 ° C. for 3 hours.
Then, the obtained reaction product was washed with 0.0
Centrifugation and washing were performed using 1M-borate buffer solution (pH 8.2), and the solid content concentration was adjusted to 5% by weight with the 0.01M-borate buffer solution, and a low-sensitivity antibody-bound colored latex particle suspension was prepared. Got

The difference in the detection sensitivity of the above antibody to Escherichia coli O157 was determined by measurement by immunochromatography.
The difference in detection sensitivity between the high-sensitivity antibody and the low-sensitivity antibody for Escherichia coli O157 was 100-fold.

3) Solution Containing Labeled Complex The suspensions of the two types of antibody-bonded colored latex particles obtained in 2) above were combined in the following manner to prepare a buffer solution [0.2M NH ₄ Cl]. , 0.9 wt% NaCl,
5% by weight choline chloride, 1% by weight sucrose (pH 8.
0)] was diluted to 0.01% by weight to obtain a solution containing the labeled complexes A to C.

Labeled complex A: High-sensitivity antibody-bound colored latex particles and low-sensitivity antibody-bound colored latex particles are mixed at a weight ratio of 2: 1 Labeled complex B: High-sensitivity antibody-bound colored latex particles only Labeled complex C: Low Sensitive antibody-bound colored latex particles only

Preparation Example 2: Preparation of immunochromatographic test piece Nitrocellulose membrane shown in FIG. 1 (manufactured by Whatman, pore size 8 μm, thickness 200 μm, 6 × 4)
0mm) goat anti-E. Coli O1 20mm from one end
57: H7 polyclonal antibody [1 mg / ml, 0.1
1.5 μL of M phosphate buffer (pH 7.4)] was applied in a line (width 1 mm) using a dispenser to obtain a membrane on which stationary phase 3 was arranged. Furthermore, 1.5 μL of rabbit anti-goat IgG polyclonal antibody [KPL, 1 mg / m1, 0.1 M-phosphate buffer solution (pH 7.4)] was placed in a line 10 mm downstream from the stationary phase 3 using a dispenser. (Width 1mm), apply reference phase 4
A membrane in which was arranged was obtained.

The obtained membrane was bovine serum albumin (manufactured by Oriental Yeast Co., 1% by weight), polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd., 0.1% by weight), and sucrose ( Wako Pure Chemical Industries, Ltd., 0.1% by weight) was immersed in an aqueous solution containing 10 minutes, and dried at 40 ° C. for 2 hours. Then, a polyester film (100 μm thick) was attached to the back side of the obtained membrane (the side opposite to the antibody-coated surface) using spray glue.

Polyester non-woven fabric Hibon 4880C (manufactured by Fuji Textile Materials Industry Co., Ltd., 6 × 7) at two points 0 to 7 mm and 9 to 16 mm from the upstream end of the nitrocellulose membrane.
mm, thickness 2.5 mm) were adhered to each other to prepare a labeled complex receiving part 1 and a test sample receiving part 2, respectively. Further, as the water absorbent pad 5, a water absorbent material made of glass fiber (10 ×
(10 mm, thickness 5 mm) was bonded in the downstream direction from a location 5 to 10 mm from the downstream end of the nitrocellulose membrane to prepare a test piece. The obtained test piece was used for the following detection.

Test Method 50 μL of the test sample was dropped on the test sample receiving part 2 of FIG.
Then, 100 μL of the solution containing the labeled complex was added dropwise to the labeled complex receiving section 1. After 15 minutes, the presence or absence of color development in the stationary phase 3 and the reference phase 4 was observed. The blue color was defined as positive.

The criteria for judgment are as follows. ++: Very strong color +: Strong color ±: Weak color -: No color developed

Test Example 1: Detection of E. coli O157 heat-treated bacterium E. coli O157 (ATCC strain 35150) was used as a test sample.
) A 10-fold serial dilution series of the heat-treated bacterium prepared with physiological saline was used. The solution containing the labeled complex is
Among those obtained in 3) of Preparation Example 1, labeled complex A (Example 1), labeled complex B (Comparative Example 1) or labeled complex C (Comparative Example 2) was used, respectively. By the above test method, the presence or absence of color development in the stationary phase 3 and the reference phase 4 was observed. The results are shown in Table 1.

[0066]

[Table 1]

In Example 1, 10 ⁴ to 10 ⁹ cells / mL
Of Escherichia coli O157 was detectable in a wide range, and the color development of reference phase 4 was clear over the entire measurement range. In Comparative Example 1, E. coli O1 was converted from 10 ⁴ cells / mL.
57 could be detected. However, at 10 ⁸ cells / mL, color development was observed in stationary phase 3, but color development in reference phase 4 was not observed, and it was not possible to make a determination. Furthermore, 10 ⁹ cells /
In mL, neither stationary phase 3 nor reference phase 4 developed color, and it was not possible to make a judgment. In Comparative Example 2, 10 ⁶ cells
E. coli could not be measured until it reached s / mL. Thus, when two kinds of labeled complexes having antibodies having different detection sensitivities to Escherichia coli O157 are used, it becomes possible to detect E. coli O157 at low to high concentrations. Further, since the color development of the reference phase 4 becomes clear over the entire measurement range, even if the test sample is E. coli O157 with high concentration,
The spreadability of the labeled complex on Escherichia coli O157, which is the test substance, can be directly confirmed, and it can be confirmed that the test was performed normally.

Test Example 2: Detection of Escherichia coli O157 from Escherichia coli O157 culture medium Escherichia coli O157 (ATCC strain 35150) was used as a test sample.
, 700375, 700376, 700377) was cultured in a TSB medium, and the obtained culture solution was used. For the number of bacteria, prepare a dilution series of the obtained culture solution and use a Petrifilm (3M) to measure 3
After culturing at 7 ° C. for 18 hours, colonies were counted and determined. As the solution containing the labeled complex, the labeled complex A (Example 2) or the labeled complex B (Comparative Example 3) among those obtained in 3) of Preparation Example 1 was used. By the above test method, the presence or absence of color development in the stationary phase 3 and the reference phase 4 was observed. The results are shown in Table 2.

[0069]

[Table 2]

In Example 2, it was confirmed that the reference phase 4 strongly developed color in all strains and that the inspection was performed normally. On the other hand, in Comparative Example 3, Escherichia coli O157
In ATCC strain 35150, 700376, and 700377, color development of reference phase 4 was not recognized, and it could not be confirmed that the inspection was normally performed. Therefore, E. coli O15
It can be seen that by using two types of labeled complexes having antibodies with different detection sensitivities to 7, the reference phase 4 is strongly colored, and it can be confirmed that the test is normally performed.

Test Example 3: Escherichia coli O15 from food culture solution
Detection of E. coli O157 (ATCC strain 35150, 700375, 70037)
6, 700377) was cultured in TSB medium. As a control, a non-inoculated E. coli was cultured in TSB medium under the same conditions. The obtained culture solution was diluted with physiological saline, 0.4 mL of a 10 ⁷ -fold diluted solution was added to 10 g of ground beef, and novobiocin (20 mg / L) -containing mEC (manufactured by Nissui Pharmaceutical Co., Ltd.) 90 m
L was added. Homogenize this with a stomacher, 4
What was statically cultured at 2 ° C. for 24 hours was used as a test sample. As the solution containing the labeled complex, the labeled complex A (Example 3) or the labeled complex B (Comparative Example 4) of the solutions obtained in Preparation Example 1 3) was used. By the above test method, the presence or absence of color development in the stationary phase 3 and the reference phase 4 was observed. The results are shown in Table 3.

[0072]

[Table 3]

In Example 3, stationary phase 3 was strongly colored in any of the strain-inoculated ground beef culture solutions, indicating that Escherichia coli O157 was present. Further, the reference phase 4 also developed color, confirming that the inspection was performed normally. In Comparative Example 4, Escherichia coli O157 ATCC strain 351
In 50, 700376, and 700377, color development was observed in all of the stationary phases 3 and O157 was detected, but no color development was observed in the reference phase 4, and it could not be confirmed that the test was normally performed. Therefore, by using two types of labeled complexes having antibodies having different detection sensitivities to Escherichia coli O157, the color development of the reference phase 4 can be ensured, and it can be confirmed that the test was performed normally. It can be seen that measurement can be performed without problems even when there is.

[0074]

EFFECT OF THE INVENTION By using the labeled complex composition for immunochromatography of the present invention, it is possible to perform highly reliable immunochromatographic measurement in a wide range of amounts of test substances.

[Brief description of drawings]

FIG. 1 is a schematic view (plan view) showing one embodiment of a test piece for immunochromatography of the present invention.

[Explanation of symbols]

1 Label complex receiving section 2 Test sample receiving section 3 stationary phase 4 Reference phase 5 Water absorption pad 6 Water-absorbent substrate

Continued front page    (72) Inventor Shuji Senda             1-1-2 Shimohozumi, Ibaraki City, Osaka Prefecture Nitto Denko             Within the corporation

Claims (8)

[Claims] 1. A labeled complex composition comprising two or more labeled complexes having a labeled substance and an antibody against the test substance, wherein the detection sensitivities of the antibodies for the labeled substance in each labeled complex are different from each other. A labeled complex composition for immunochromatography, comprising: 2. The labeled complex composition according to claim 1, wherein the difference between the maximum detection sensitivity and the minimum detection sensitivity is a 5 to 5 × 10 ⁴ times difference measured by immunochromatography. 3. The labeled complex composition according to claim 1, wherein the labeling substance is colored particles or an enzyme. 4. The labeled complex composition according to claim 1, wherein the labeled complex is obtained by fixing a labeling substance and an antibody on metal colloid particles or water-dispersed polymer particles. 5. Using a test piece having a stationary phase in which an antibody against a test substance is immobilized on a water-absorbent substrate, the presence or absence of the test substance in the test sample is determined as follows (a). b): immunity, characterized by being detected by the presence or absence of a complex with (a) a test substance and (b) a labeled complex in the labeled complex composition according to any one of claims 1 to 4. Chromatographic method. 6. The test piece has a reference phase on which a substance capable of specifically binding to an antibody contained in a labeled complex is immobilized on a water-absorbent substrate downstream of the stationary phase. The immunochromatographic method according to claim 5. 7. The immunochromatographic method according to claim 5, further comprising the step of dropping a solution containing the labeled complex composition onto a test piece to bring it into contact with a test sample. 8. The test piece has a labeled complex phase in which two or more labeled complexes according to any one of claims 1 to 4 are detachably retained by contact with a test sample. The immunochromatographic method according to claim 5 or 6.